Abstract

A simple aqueous extraction method for the isolation and identification of psilocin from Psilocybe Cubensis
mushrooms is reported. This method employs a dephosphorylation of the phosphate ester to psilocin, which
facilitates a greater product yield and simplifies identification. Psilocin extracted by this method is sufficiently
concentrated and free of co-contaminants to allow identification by infrared spectroscopy and gas chromatography/mass spectrometry.

The tryptamines are one of four categories of hallucinogenic indoles in more than 20 classes of indole
compounds comprising approximately 600 alkaloids1. Considerable research has been conducted with psilocin and
psilocybin since their isolation by Hofmann et. al.2 Several extraction techniques1,3-6 have been used to
isolate psilocin and psilocybin from more than two dozen species of mushrooms in four genera (Conocybe.
Panaeolus, Psilocybe, Stropharia). The techniques that use methanol co-extract other compounds such as urea,
ergosterol, ergosteral peroxide, α,α-trehalose, baeocystin, and norbaeocystin3,4,7. At present, a useful
aqueous extraction procedure has not been reported for psilocin and psilocybin.

The dephosphorylation of psilocybin to psilocin in vivo has been well documented1,8,9 and is thought to
account for most or all of its central nervous system activity8. Conversion of psilocybin to psilocin is also
necessary for aqueous extraction with organic solvents because of the very low lipid solubility of psilocybin.
Extraction of only one compound also permits infrared analysis of the extract.

Concentration and detectability of psilocin and psilocybin are dependent on several variables, including:

The absence of glucose, which will prevent the production of psilocybin10.

Low levels of ammonium succinate, which will give poor yields of psilocybin10.

Timing: maximum production of psilocybin occurs on the seventh day after germination, while maximum production of the mycelium is reached by the ninth day10.

Temperature: complete loss of psilocin and psilocybin will occur in harvested mushrooms left at room temperature for an extended period of time3.

Oxidation: psilocin will oxidize to a blue product (possibly accounting for the bluing color in the four genera containing psilocin and psilocybin)9.

Because of the increasing popularity of these mushrooms and kits available from drug oriented publications
for growing mushrooms containing psilocin and psilocybin in cow manure a simple aqueous extraction procedure
has been developed that extracts reasonably pure psilocin from mature mushrooms. This extraction method
greatly simplifies the identification of psilocin from those mushrooms by infrared spectroscopy and gas
chromatography/mass spectrometry (GS/MS).

Experimental

A representative sample of 2 to 10g of dried mushrooms is ground to a fine powder by mortar and pestle. The
powder is mixed with 100 mL of dilute acetic acid in a 250-mL beaker. The pH is readjusted to pH 4 with
glacial acetic acid. After standing 1 h, the beaker is placed in a boiling water bath for 8 to 10 min or until
the internal temperature of the acid mixture reaches 70°C. The beaker is removed and cooled to room
temperature under running water. The acid mixture is separated from the mushroom powder by suction filtration
using glass wool. The filtrate is brought to pH 8 with concentrated ammonium hydroxide and quickly extracted
with two 50-mL portions of diethyl ether. Gentle mixing instead of shaking should be used to prevent an
emulsion. The ether is dried over sodium sulfate, filtered, and evaporated under nitrogen with no applied heat.

Crude psilocin will appear as a greenish residue. Recrystallization from chloroform/heptane (1:3) yields
white crystals. The resulting powder can then be submitted to infrared and mass spectral analyses.

Results and Discussion

This method permits rapid isolation of psilocin from hallucinogenic mushrooms by co-extraction of both
psilocin and psilocybin. Dilute acetic acid is an excellent solvent for this purpose, because both compounds
are very soluble in acetic acid11 and very little of other interfering substances are extracted, It is most
likely some other compounds are co-extracted but are removed from psilocin in the ether extraction from the
aqueous base. Psilocybin is completely dephosphorytated to psilocin by heating the acid extract. After
addition of the base, extraction into ether should be performed promptly, because of decomposition of psilocin
at a greater pH than 712. The extraction and dephosporylation steps produce reasonably pure psilocin from a
small amount of mushroom material. Two grams of mushrooms will often be sufficient to obtain an infrared
spectrum of psilocin (Fig. 1). Smaller mushrooms exhibits provide ample psilocin for mass spectral
analysis (Fig. 2).

This method has been used in our laboratory for six months and has given excellent results in separating
psilocin from methanol-soluble compounds. Other identification techniques such as gas chromatography and
microcrystalline tests are possible on psilocin extracted by this method.